Herein, we demonstrate that the ubiquitin-associated (UBA) domain of sequestosome 1/p62 displays a preference for binding K63-polyubiquitinated substrates. Furthermore, the UBA domain of p62 was necessary for aggregate sequestration and cell survival. However, the inhibition of proteasome function compromised survival in cells with aggregates. Mutational analysis of the UBA domain reveals that the conserved hydrophobic patch MGF as well as the conserved leucine in helix 2 are necessary for binding polyubiquitinated proteins and for sequestration-aggregate formation. We report that p62 interacts with the proteasome by pull-down assay, coimmunoprecipitation, and colocalization. Depletion of p62 levels results in an inhibition of ubiquitin proteasome-mediated degradation and an accumulation of ubiquitinated proteins. Altogether, our results support the hypothesis that p62 may act as a critical ubiquitin chain-targeting factor that shuttles substrates for proteasomal degradation.Sequestosome 1/p62 is a cellular protein which was initially identified as a phosphotyrosine-independent ligand of the Src homology 2 (SH2) domain of p56 lck (27). The protein has been cloned by two independent groups as a cointeracting protein of the atypical protein kinase C (aPKC) and is also named ZIP for PKC-interacting protein (47, 50). p62 has been shown to bind ubiquitin noncovalently (56), and its overexpression results in large cytoplasmic aggregates (47). We have recently determined that p62 possesses sequence homology with other proteins possessing a ubiquitin-associated (UBA) domain at their C terminus, amino acids 386 to 434 (16), in p62. Interestingly, p62 contains several structural motifs which suggest that it might participate in the formation of multimeric signaling complexes. These domains include an acidic interaction domain (AID/ORCA/PC/PB1) that binds the aPKC, a ZZ finger, a binding site for the RING finger protein TRAF6, two PEST sequences, and the UBA domain (16).Ubiquitin is a small polypeptide of 76 amino acids that can be covalently attached to other proteins. Monoubiquitination serves as a novel endocytosis signal (19), whereas polyubiquitin chains target substrates for degradation by the proteasome (45). p62 has been shown to interact in a noncovalent fashion with polyubiquitin chains (5, 53), which is consistent with reports demonstrating that proteins possessing UBA domains are more likely to bind polyubiquitin chains over monoubiquitin (59). Conjugation of ubiquitin to substrate proteins requires three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Initially, E1 activates ubiquitin by forming a high-energy thioester intermediate with the C-terminal glycine using ATP. The activated ubiquitin is sequentially transferred to E2 and then to E3 which catalyzes isopeptide bond formation between the activated C-terminal glycine of ubiquitin and an ε-amino group of a lysine residue in the substrate (45). However, only HECT-type E3s can form thioester bon...

Nerve growth factor (NGF) binding to p75(NTR) influences TrkA signaling, yet the molecular mechanism is unknown. We observe that NGF stimulates TrkA polyubiquitination, which was attenuated in p75(-/-) mouse brain. TrkA is a substrate of tumor necrosis factor receptor-associated factor 6 (TRAF6), and expression of K63R mutant ubiquitin or an absence of TRAF6 abrogated TrkA polyubiquitination and internalization. NGF stimulated formation of a TrkA/p75(NTR) complex through the p62 scaffold, recruiting the E3/TRAF6 and E2/UbcH7. Peptide targeted to the TRAF6 binding site present in p62 blocked interaction with TRAF6 and inhibited ubiquitination of TrkA, signaling, internalization, and NGF-dependent neurite outgrowth. Mutation of K485 to R blocked TRAF6 and NGF-dependent polyubiquitination of TrkA, resulting in retention of the receptor on the membrane and an absence in activation of specific signaling pathways. These findings reveal that polyubiquitination serves as a common platform for the control of receptor internalization and signaling.

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Inclusions isolated from several neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by ubiquitin-positive proteinaceous aggregates. Employing confocal and immunoelectron microscopy, we find that the ubiquitin-associating protein sequestosome1/p62, co-localizes to aggregates isolated from AD but not control brain, along with the E3 ubiquitin ligase, TRAF6. This interaction could be recapitulated by co-transfection in HEK293 cells. Employing both in vitro and in vivo approaches, tau was found to be a substrate of the TRAF6, possessing lysine 63 polyubiquitin chains. Moreover, tau recovered from brain of TRAF6 knockout mice, compared with wild type, was not ubiquitinated. Tau degradation took place through the ubiquitinproteasome pathway and was dependent upon either the K63-polyubiquitin chains or upon p62. In brain lysates of p62 knockout mice, tau fails to co-interact with Rpt1, a proteasomal subunit, thereby indicating a requirement for p62 shuttling of tau to the proteasome. Our results demonstrate that p62 interacts with K63-polyubiquitinated tau through its UBA domain and serves a novel role in regulating tau proteasomal degradation. We propose a model whereby either a decline in p62 expression or a decrease in proteasome activity may contribute to accumulation of insoluble/aggregated K63-polyubiquitinated tau.

Sequestosome 1/p62 is a scaffolding protein with several interaction modules that include a PB1 dimerization domain, a TRAF6 (tumor necrosis factor receptor-associated factor 6) binding site, and a ubiquitin-associating (UBA) domain. Here, we report that p62 functions to facilitate K63-polyubiquitination of TRAF6 and thereby mediates nerve growth factor-induced activation of the NF-B pathway. In brain of p62 knock-out mice we did not recover polyubiquitinated TRAF6. The UBA domain binds polyubiquitin chains and deletion of p62-UBA domain or mutation of F406V within the ubiquitin binding pocket of the UBA domain abolished TRAF6 polyubiquitination. Likewise, deletion of p62 N-terminal dimerization domain or the TRAF6 binding site had similar effects on both polyubiquitination and oligomerization of TRAF6. Nerve growth factor treatment of PC12 cells induced TRAF6 polyubiquitination along with formation of a p62-TRAF6-IKK␤-PKC signal complex, while inhibition of the p62/TRAF6 interaction had an opposite effect. These results provide evidence for a mechanism whereby p62 serves to regulate the NF-B pathway. Nuclear Factor B (NF-B)2 plays an important role in the regulation of a number of genes involved in neuronal development and survival, as well as numerous other cellular functions, such as inflammation, immunity, and cellular differentiation (1, 2). Constitutive NF-B activity is necessary for neuronal survival (3) and neurite outgrowth (4). In its inactive state NF-B is bound by its inhibitor protein, IB, in the cytoplasm (1). Once a cell is activated, IB is phosphorylated by IB kinase (IKK) and tagged by ubiquitin for proteasomal degradation, freeing NF-B to enter the nucleus (2). Nerve growth factor (NGF) elicits its biological response by binding to two receptors, TrkA and p75. The p75 receptor activates NF-B by binding and interacting directly with TRAF6 (5) and a receptor-associated kinase, IRAK (interleukin receptor-associated kinase) (6). Through an adaptor protein, p62, the p75 receptor connects with the atypical protein kinase Cs (aPKCs), which serve as upstream kinases for phosphorylation and activation of IKK␤ (7) in NGF and IL-1 signaling (8), suggesting a common and conserved function for p62 in both the immune and nervous systems.Sequestosome 1/p62 was cloned as the interacting protein of the atypical PKCs (9, 10) and serves as a scaffold comprised of numerous protein interaction modules that allow effector proteins to converge and interact with their substrates, thus passing the signal downstream to activate 12). Both the aPKCs, and , and tumor necrosis factor receptor-associated factor (TRAF6) specifically interact with p62; aPKC interacts with the PB1 domain (amino acids 3-100), and TRAF6 interacts with the TRAF6 binding domain (amino acids 228 -254) (12). The interaction of p62 and TRAF family members is specific to p62 and TRAF6, as neither TRAF5 nor TRAF2 interact with p62 (8). The N terminus of p62 is also necessary for p62 dimerization (13), and p62 also possesses a UBA domain at its C terminu...

Nerve growth factor (NGF) binding to both p75 and TrkA neurotrophin receptors activates the transcription factor nuclear factor B (NF-B). Here we show that the atypical protein kinase C-interacting protein, p62, which binds TRAF6, selectively interacts with TrkA but not p75. In contrast, TRAF6 interacts with p75 but not TrkA. We demonstrate the formation of a TRAF6-p62 complex that serves as a bridge linking both p75 and TrkA signaling. Of functional relevance, transfection of antisense p62-enhanced p75-mediated cell death and diminished NGF-induced differentiation occur through a mechanism involving inhibition of IKK activity. These findings reveal a new function for p62 as a common platform for communication of both p75-TRAF6 and TrkA signals. Moreover, we demonstrated that p62 serves as a scaffold for activation of the NF-B pathway, which mediates NGF survival and differentiation responses.

Several highly conserved p62 homologs have recently been isolated, e.g. the rat atypical protein kinase C-interacting protein (ZIP), the murine A170/signal transduction and adapter protein, and the human p62, a protein that binds the Src homology 2 domain of p56 lck . These proteins share striking similarity in amino acid sequence and structural motifs, thereby suggesting conserved functional properties. ZIP/p62 has been shown to play an important role as a scaffold leading to the activation of the transcription factor nuclear factor U UB. In addition, a nuclear form of p62 has been characterized that can serve as a transcriptional co-activator. Moreover, p62 is capable of binding ubiquitin (Ub) non-covalently through its Ubassociated domain. In this review, we will focus on the structure and function of ZIP/p62. ß

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